Interleukin 15 mediates epithelial changes in celiac disease Luigi Maiuri, Carolina Ciacci, Salvatore Auricchio, Virginia Brown, Sonia Quaratino, Marco Londei  Gastroenterology  Volume 119, Issue 4, Pages 996-1006 (October 2000) DOI: 10.1053/gast.2000.18149 Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 1 Effect of IL-15 on TFR antigen expression by enterocytes of treated celiac disease small intestinal biopsy specimens after 24 hours of in vitro challenge. Expression of TFR in enterocytes after a 24-hour in vitro challenge of treated celiac disease specimens with or without IL-15 (n = 14), IL-7 (n = 7), IL-4 (n = 7), IL-2 (n = 7), or gliadin (n = 14). Open bars show low expression (0/1+) in >70% enterocytes; filled bars show high expression (2+) in >70% enterocytes. *P < 0.01 vs. cultures with medium alone. Gastroenterology 2000 119, 996-1006DOI: (10.1053/gast.2000.18149) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 2 Effect of IL-15 on TFR antigen expression by enterocytes of treated celiac disease small intestinal biopsy specimens after 24 hours of in vitro challenge. (A) Expression of TFR after a 24-hour challenge of treated celiac disease biopsy specimens with medium alone. Weak staining is observed in some crypt enterocytes. (B) Expression of TFR after a 24-hour challenge with IL-15. Intense staining is observed in the cytoplasm of crypt enterocytes. (Peroxidase staining technique; original magnification 180×.) Gastroenterology 2000 119, 996-1006DOI: (10.1053/gast.2000.18149) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 3 Effect of IL-15 on Ki67 antigen expression by enterocytes of treated celiac disease small intestinal biopsy specimens after 24 hours of in vitro challenge. Expression of enterocyte Ki67 after a 24-hour in vitro challenge of treated celiac disease specimens with or without IL-15 (n = 10), IL-7 (n = 7), IL-4 (n = 7), IL-2 (n = 7), or gliadin (n = 10). *P < 0.0001; °P < 0.05 vs. cultures with medium alone. Gastroenterology 2000 119, 996-1006DOI: (10.1053/gast.2000.18149) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 4 Effect of IL-15 on Ki67 antigen expression by enterocytes of treated celiac disease small intestinal biopsy specimens after 24 hours of in vitro challenge. (A) Expression of Ki67 after a 24-hour challenge of treated celiac disease specimens with medium alone. Expression of Ki67 is observed in a few crypt enterocytes. (B) Expression of Ki67 after a 24-hour challenge of treated celiac disease specimens with IL-15. Ki67 nuclear expression is evident in many crypt enterocytes. (Peroxidase staining technique; original magnification 180×.) Gastroenterology 2000 119, 996-1006DOI: (10.1053/gast.2000.18149) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 5 Effect of IL-15 on FAS expression by enterocytes of treated celiac disease small intestinal biopsy specimens after 24 hours of in vitro challenge. Expression of FAS in enterocytes after a 24-hour in vitro challenge of treated celiac disease specimens with or without IL-15 (n = 14), IL-7 (n = 7), IL-4 (n = 7), IL-2 (n = 7), or gliadin (n = 14). Open bars show low expression (0/1+) in >70% enterocytes; filled bars show high expression (2+) in >70% enterocytes. *P < 0.01 vs. cultures with medium alone. Gastroenterology 2000 119, 996-1006DOI: (10.1053/gast.2000.18149) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 6 Effect of IL-15 on FAS expression by enterocytes of treated celiac disease small intestinal biopsy specimens after 24 hours of in vitro challenge. (A) FAS expression after a 24-hour challenge of treated celiac disease specimens with medium alone. Very faint staining is observed on basolateral cell membranes of a few enterocytes. (B) FAS expression after 24 hours of challenge of treated celiac disease biopsy specimens with IL-15. Intense FAS expression is evident on basolateral cell membranes of most enterocytes and in some LPMNCs. (Peroxidase staining technique; original magnification 160×.) Gastroenterology 2000 119, 996-1006DOI: (10.1053/gast.2000.18149) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 7 Effect of neutralizing anti–IL-15 M110 mAb on epithelial modifications induced by gliadin challenge after 24 hours of in vitro culture of treated celiac disease small intestinal biopsy specimens. (A) Effect of M110 mAb and of isotype-matched control mAbs (anti-human lactase mlac1 and anti-CD80 BB7.1 mAbs; n = 5) on gliadin-induced TFR enterocyte expression. Open bars show low expression (0/1+) in >70% enterocytes; filled bars show high expression (2+) in >70% enterocytes. °P = 0.003 vs. cultures with medium alone; *P = 0.02 vs. cultures with PT gliadin digest. (B) Effect of M110 mAb and of isotype-matched control mAbs (antihuman lactase mlac1 and anti-CD80 BB7.1 mAbs; n = 5) on gliadin-induced FAS enterocyte expression. Open bars show low expression (0/1+) in >70% enterocytes; filled bars show high expression (2+) in >70% enterocytes. °P = 0.003 vs. cultures with medium alone; *P = 0.02 vs. cultures with PT gliadin digest. Gastroenterology 2000 119, 996-1006DOI: (10.1053/gast.2000.18149) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 8 Cultured untreated celiac disease biopsy specimens: effect of IL-15 on enterocyte DNA fragmentation and neutralizing effect of anti–IL-15 M110 mAb on gliadin-induced enterocyte DNA fragmentation. (A) Effect of IL-15 and gliadin on enterocyte DNA fragmentation after a 24-hour challenge of untreated celiac disease specimens with villous atrophy (n = 4). *P < 0.05 vs. cultures with medium alone. (B) Effect of M110 mAb and of isotype-matched control mAbs (anti-human lactase mlac1 and anti-CD80 BB7.1 mAbs) on gliadin-induced enterocyte DNA fragmentation after 24-hour challenge of untreated celiac disease biopsy specimens with villous atrophy (n = 5). °P < 0.01 vs. cultures with medium alone; *P < 0.01 vs. cultures with PT gliadin digest. Gastroenterology 2000 119, 996-1006DOI: (10.1053/gast.2000.18149) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 9 (A) Pattern of enterocyte DNA fragmentation after a 24-hour gliadin challenge of untreated celiac disease specimens with villous atrophy. Many enterocytes are TUNEL positive; the staining is also evident in LPMNCs. (B) Pattern of DNA fragmentation after a 24-hour in vitro challenge of untreated celiac disease specimens with gliadin supplemented with neutralizing anti–IL-15 M110 mAb. A marked reduction in the number of TUNEL-positive enterocytes is observed with respect to the pattern observed after gliadin challenge. (TUNEL technique; original magnification 160×.) Gastroenterology 2000 119, 996-1006DOI: (10.1053/gast.2000.18149) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 10 IL-15 competitive PCR in celiac disease and control small intestine. Competitive RT-PCR using RNA isolated from intestinal biopsy specimens from 3 patients and 3 controls. Total RNA (2 μg) was used to generate cDNA in an RT reaction. Next, 2 μL of cDNA was amplified in a competitive PCR using decreasing concentrations of the competitor template. Endogenously amplified IL-15 migrates below the competitor in the agarose gel. The amounts of competitor DNA added to each PCR reaction were: 2 × 10−2, 2 × 10−3, 2 × 10−4, 2 × 10−5, and 2 × 10−6 ng. Amplification of β-actin served as control to normalize the amount of cDNA input in the competitive PCR reaction. Gastroenterology 2000 119, 996-1006DOI: (10.1053/gast.2000.18149) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 11 IL-15 protein expression in celiac disease and control small intestine. Expression of IL-15 by LPMNCs of untreated celiac (n = 14), treated celiac (n = 14), and control (n = 10) intestine (mean ± SD). *P < 0.005 vs. treated celiac disease and control samples. Gastroenterology 2000 119, 996-1006DOI: (10.1053/gast.2000.18149) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 12 IL-15 protein expression in celiac disease and control small intestine. (A) Expression of IL-15 in untreated celiac disease intestine. Some LPMNCs are clearly stained (red) in untreated celiac disease duodenum. (B) Expression of IL-15 in treated celiac disease intestine. No detectable staining is found in the lamina propria region shown. Only a very few LPMNCs are IL-15 positive when 1 mm2 of lamina propria is analyzed. (Alkaline phosphatase staining technique; original magnification 160×.) Gastroenterology 2000 119, 996-1006DOI: (10.1053/gast.2000.18149) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 13 IL-15 protein expression after 3 hours of in vitro challenge of treated celiac disease small intestinal biopsy specimens with PT gliadin digest. Expression of IL-15 by LPMNCs of treated celiac disease intestine after a 3-hour in vitro culture with or without PT gliadin digest (n = 5; mean ± SD). *P < 0.005 vs. samples cultured with medium alone. Gastroenterology 2000 119, 996-1006DOI: (10.1053/gast.2000.18149) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 14 IL-15 protein expression after 3 hours of in vitro challenge of treated celiac disease small intestinal biopsy specimens with PT gliadin digest. (A) Three-hour in vitro organ culture of treated celiac disease specimens with medium alone. Only a very few IL-15–positive cells are detected (arrow); this pattern is similar to that observed before in vitro manipulation. (B) Three-hour in vitro challenge of treated celiac disease specimens with PT gliadin digest. Numerous IL-15–positive LPMNCs can be seen (arrow). (C) Three-hour in vitro challenge of treated celiac disease specimens with PT gliadin digest: distribution of IL-15 protein expression. IL-15–positive cells detected have the phenotype of resident macrophage/monocytes. Two-color immunofluorescence with anti–IL-15 (red) and CD68 (green) mAbs (see Materials and Methods). Note that some cells (5 in the present field) show colocalization of both markers (orange/yellow). Some CD68+ cells do not express IL-15 (green). (Immunofluorescence; original magnification 150× [A and B] and 260× [C].) Gastroenterology 2000 119, 996-1006DOI: (10.1053/gast.2000.18149) Copyright © 2000 American Gastroenterological Association Terms and Conditions